This application seeks support for a project of research directed toward better understanding of the molecular mechanisms and cellular functions of ion transport across human red blood cell membranes. The overall goal of this grant project is the development and use of patch clamp techniques to answer four previously unanswered questions about the structure and functions of human red blood cell membranes. 1. Are there cryptic voltage-dependent ion channels in human red cells? If so, what are their single channel characteristics? 2. Are the kinetics of ionophore-mediated transport the same in cell membranes as in artificial planar lipid bilayers? 3. What is the net surface charge density on the cytoplasmic and external surfaces of the red cell membrane? What is the dipole potential difference that is experienced by carrier-transported ions? 4. What are the kinetic characteristics of the directly measured chloride conductance of human red cells? Can the kinetics of rapid anion exchange and low anion conductance be reconciled in a common molecular mechanism, band 3? Additionally, we will continue to investigate the mechanisms of co-transport and test the hypothesis that the ion pair between sodium and carbonate is a monovalent anion substrate for the anion exchange mechanism. Experiments are designed to answer questions relevant to these problems. They involve observations of net and tracer fluxes of inorganic ions and electrical measurements of patch clamped native and ionophore-treated human red cells in media of different ion compositions and different total ionic strengths.